Project Summary/Abstract The proposal seeks to develop ultra-sensitive methods for structure elucidation of natural products (NPs) to accelerate discovery and structure elucidation of rare marine natural products as leads for drug development for therapeutic applications in infectious disease and cancer. New biologically active NPs have long been resources for drug discovery; a historical legacy that reflects their unique pharmacology, success in generating leads that advance into clinical trials, and approval as new drugs (e.g. Taxol, Yondelis). Modern drug discovery from NPs, under current technological limitations, have resulted in diminishing returns of new chemical entities against a rising rate of re-isolation of known NPs; usually the principal components. The ?low-hanging fruit? problem is particularly severe in discovery of natural products from bacteria, where the work-flow dedicated to dereplication has become outsized relative to rate of discovery of NCEs. Screening of marine invertebrate extracts, with their typically complex microbial flora, passes over NPs present at vanishingly low concentrations because of inherent biases of high-throughput screening (HTS) approaches. We proposed an integrated methodology, which combines MS-based discovery of new natural products (NPs) from niche sources through a deep chemical diversity ?bottom up (DCD-BU) approach that innovatively mines 'nanomole-scale' chemical diversity beyond the dynamic range of conventional natural products isolation-discovery approaches, and close to the limits of detection. The enabling technology involves integration and refinement of 'nanomole-scale' methods using microcryoprobe high-field NMR, that is 20x more sensitive than conventional instrumentation, and CD-based tools for investigation of NP absolute configuration. In this pilot project, the DCD-BU will be applied to a small panel of non-cultureable niche marine organisms: rare, encrusting ?veneer? sponges, including Halisarca and Spirastrella, that are chemically-rich, but underexplored. A new method for assignment of absolute stereostructures of glycol NPs, including tertiary alcohols, using exciton coupled circular dichroism of 3- methylthioacrylate diesters, has been developed and will be applied to a panel of model compounds, then extended to new NPs isolated by DCD-BU.